CH637218A5 - POLYMER MATRIX BASED ON ACRYLAMIDE. - Google Patents

Description

The object of the invention is therefore to provide a polymer matrix with which the binding specificity of the antibody to be enclosed can be increased and undesired cross-reactions can be prevented.

Surprisingly, it has been shown that a variation and increase in the binding specificity of the antibody can be achieved by polymer matrices, which are copolymers of acrylamide.

5 The invention relates to a polymer matrix based on acrylamide for immobilizing antibodies in the radioimmunological determination of hormones and pharmaceuticals, which is characterized in that it consists of a copolymer of acrylamide.

10 Copolymers of acrylamide and one or more of the compounds acrylic acid, methacrylic acid, methacrylamide, their derivatives and salts of acrylic acid or methacrylic acid are particularly preferred.

By copolymerizing the acrylamide with 15 copolymerizable compounds, the microenvironment of the polymer matrix can be influenced. The effect is based essentially on hydrophobic and hydrophilic as well as electrostatic effects.

By varying the polymer matrix by means of copolymerization, it is possible to significantly increase the binding specificity of the antibody and to suppress undesired cross-reactivities. The polymer matrix is varied by suitable copolymerization, on the one hand to ensure a specific reaction of the haptens with the immobilized antibody and on the other hand to prevent cross-reacting haptens.

Due to the increased specificity, the polymer matrices according to the invention make it possible to measure directly in the non-extracted serum.

3o With the aid of the polymer matrices according to the invention, a very precise radioimmunological determination of hormones and pharmaceuticals is possible, since the fluctuation of the determination values due to unspecific bonds between the steroid molecules and the matrix is minimized by the copolymerization.

With various provisions such as those of the thyroid hormones thyroxine and triiodothyronine are used, such as anilinonaphtholsulfonic acid, merthiolate or salicylates, to displace the hormones from the binding 40 with the various binding proteins in serum. However, it is known that at higher concentrations of these competitive substances there is an impairment of the binding between the hormone and the antibody. When using the polymer matrices according to the invention, it is possible, presumably on account of electrostatic interactions, to increase the concentration of the competitive substances substantially, namely by a factor of 100, without weakening the binding of hormone-antibody.

The proportion of acrylamide in the copolymer can be between 1 and 99 mol%, preferably 5 to 95 mol% and in particular 20 to 80 mol%.

Copolymers of acrylamide and methacrylic acid are particularly suitable, and in particular those in which the 55 methacrylic acid content is 20 to 60 mol%. The methacrylic acid on the one hand makes the matrix hydrophobic and on the other hand leads to a charge effect, which largely eliminates the non-specific binding of the substances to be determined.

60 By using copolymers according to the invention which contain acrylic acid or methacrylic acid or their salts, it is also possible to achieve a neutralization effect or a buffering action for acidic or alkaline solutions. This is part of numerous provisions of essential pre-65.

The alkali and / or alkaline earth salts are preferred as salts of acrylic acid or methacrylic acid. Sodium or calcium salts are particularly suitable.

3rd

637 218

Suitable derivatives of acrylic acid and methacrylic acid are, in particular, their esters, for example the methyl and ethyl esters.

Suitable derivatives of methacrylamide are, for example, compounds substituted on nitrogen, such as N-hydro-5 xymethyl methacrylamide.

Other suitable compounds copolymerizable with acrylamide are the derivatives of acrylamide, in particular compounds substituted on nitrogen, such as N-hydroxy-methylacrylamide. io

An example of other compounds copolymerizable with acrylamide is N, N'-diallyltartardiamide.

The antibody is immobilized by influence in the polymer matrix and / or by covalent fixation on the polymer matrix. 15

A detailed description of the radioimmunoassay can be found, for example, in Clinical Chemistry, Vol. 19, No. 2, 1973, p. 145. In Clinical Chemistry, Vol. 19, No. 12, 1973, p. 1339 and Clinical Chemistry, Vol. 21 , No. 7, 1975, p. 829 describes radioimmunological techniques using an immobilized antibody.

The polymer matrices according to the invention are suitable for determining the various hormones and pharmaceuticals which are partly bound in serum or plasma to specific or non-specific binding proteins. Examples include the thyroid hormones, in particular thyroxine and triiodothyronine, the steroid hormones such as cortisol, testosterone, progesterone, estrone, estradiol and estriol and the cardiac glycosides such as digitoxin and digoxin. Vitamins, especially vitamin 30 B12 and folic acid, as well as pharmaceuticals with strong protein binding, such as anticoagulants, dicumarol, analgesics and salyzilates, can also be determined.

In addition to radioimmunological determination, alternative determination methods, such as fluoroimmunological determination or determination by means of enzymatic labeling, may also be considered.

The synthesis of the antigens, the production of antisera, for example by immunizing rabbits, and the isolation and immobilization of the antibodies are known «(see, for example, Clinical Chemistry, Vol. 19, No. 2.1973, p. 146 ff.).

The polymer matrices with the immobilized antibody can be produced, for example, in the manner

that a solution of the antibody is added to the monomer mixture 45. The batch is polymerized by free radicals, for example, and the polymer obtained is comminuted, washed and dried.

The selection and the proportions of the monomers added to the acrylamide usually depend on the desired properties of the polymer matrix with regard to the specificity. The specificity can be influenced within wide limits by changing the hydrophobicity and the charge of the matrix. The addition of methacrylamide increases the hydrophobicity. The charge of the matrix is varied by the use of acrylic acid or methacrylic acid or their salts. There is both the possibility of adding the salts of acrylic acid or methacrylic acid directly to the monomer mixture or, alternatively, the acids can be polymerized and the salts can then be formed in the polymer matrix by ion exchange.

To achieve a suitable pore size of the polymer matrix, the monomer concentration is generally varied. A monomer concentration in the range of about 20% leads to a pore size of about 7 to 10 Â. 65

An advantageous copolymer consists, for example, of acrylamide and 20 to 60 mol% of acrylic acid and / or methacrylic acid, prepared from an approximately 20% monomer solution,

wherein at least a portion of the acid groups is converted to the corresponding alkali or alkaline earth salts.

The invention is explained in more detail below with the aid of examples.

example

This example shows the influence of the polymer matrix on the cross-reactivity (K) of estriol-3-glucuronide and estriol-3-sulfate.

The cross-reactivity K is calculated from the hundred times the required mass of the corresponding hapten for y = 50 divided by the required mass of the cross-reacting hapten for y = 50. The values are calculated on the basis of ID50.

The selected anti-estriol antibody showed a high cross-reactivity of the 3-glucuronide conjugates and the 3-sulfate conjugates.

For each polymerization batch, the concentration was adjusted so that the total monomer concentration was 2.9 mol / l. For one approach, e.g. 5 g of acrylamide and 1.25 g of N, N'-methylenebisacrylamide dissolved in a beaker in 24 ml of phosphate buffer with a pH of 7.2. After the antibody had been mixed in 1 ml of phosphate buffer, the reaction was started with 0.15 mg of riboflavin and 0.10 ml of N, N, N ', N'-tramethylethylenediamine and irradiated with UV light. The temperature was kept below 50 ° C. during the irradiation time of 45 min. The gel block was then crushed, washed with distilled water and dried.

22 mg of dry anti-estriol antibody gel were weighed into small columns and 0.150 ml of incubation solution was drawn up. This incubation solution contained 3H estriol and unlabelled hapten (estriol, estriol-3-glucuronide or estriol-3-sulfate). The reaction temperature was kept constant at 0 ° C. After an incubation period of 30 min, the free from the antibody-bound hapten was separated by elution with phosphate buffer containing albumin. The eluate was collected in scintillation vials, 15 ml of scintillation liquid was added and the radioactivity was measured in the liquid scintillator. The concentration of the free indicator hapten 3H-estriol was calculated from the impulses.

The results for determinations in the aqueous system and using polymer matrices from acrylamide and acrylamide copolymers are summarized in the table below. The values given are the cross-reactivity values defined above.

aqueous acrylic-amide system

100

Sodium salt of acrylic acid / acrylamide

10:90 30:70 50:50

Sodium salt of methacrylic acid / acrylamide 20:80 40:60 60:40

55 ..

Estriol

61

29

19th

10th

14

14 4

21st

3-glucu

ronid

Estriol

47

33

30th

12

24th

27 6

23

3-sulfate

The results show that the copolymers have a significant increase in binding specificity compared to aqueous systems and acrylamide homopolymers. Appropriate selection of the copolymer can increase the specificity by a factor of 15 compared to aqueous systems and by a factor of 7 compared to acrylamide homopolymers. This shows a comparison of the values

637 218

4th

of the aqueous system and of acrylamide homopolymer with a 40/60 copolymer of methacrylic acid and acrylamide.

Example 2

This example shows the use of the polymer matrix according to the invention in the overall determination of thyroxine by enzymatic hydrolysis with pepsin.

The polymer matrix for the antibody gel consisted of a copolymer of acrylamide and 40 mol% of the sodium salt of methacrylic acid, prepared from a 20% monomer solution.

10 μl of serum with a content of 18 μg of thyroxine per 100 ml were mixed with 160 μl of an enzyme solution which consisted of 2 mg / ml of pepsin dissolved in 0.1 N hydrochloric acid.

The reaction with the enzyme was carried out at room temperature for 30 minutes.

150 μl of tracer solution containing 5.2 ng / ml of radioactively labeled thyroxine was then added to these 170 μl.

The entire solution was then applied to 60 mg of antibody gel. The incubation time was 30 minutes and the temperature was 22 ° C.

The evaluation showed an extremely high recovery of 98%.

Example 3

This example shows the use of the polymer matrix according to the invention in the overall determination of cortisol.

The polymer matrix for the antibody gel consisted of a copolymer of acrylamide and 40 mol% of one

Mixture of sodium and calcium salts of methacrylic acid, ■ made from a 20% monomer solution.

10 μl of serum or plasma with a content of 15 μl of cortisol per 100 ml were presented. 500 ml of tracer solution consisting of 125 I-cortisol in aqueous citrate buffer pH 3.5 were added.

The entire solution was then applied to 100 mg of antibody gel. The incubation time was 20 min, the temperature 25 ° C.

io. The evaluation showed an extraordinarily high recovery of 99%.

Example 4

This example shows the use of the polymer matrix according to the invention in the determination of thyroxine using competitive substances.

The polymer matrix for the antibody gel consisted of a copolymer of acrylamide and 40 mol% of a calcium-sodium mixed salt of methacrylic acid, prepared from 20% of a 20% monomer solution.

500 ml of tracer solution consisting of I25J thyroxine in phosphate buffer pH 9.6 were applied to 80 mg of antibody gel.

In addition, 500 μl of the same tracer solution, which contained 25 6 mg / ml of anilinonaphtholsulfonic acid, was applied to 80 mg of antibody gel.

Both samples were incubated at a temperature of 22 ° C for 20 min.

The evaluation showed no impairment of the binding of the thyroxine to the antibody despite the high concentration of the competitive substance present, namely anilinonaphtholsulfonic acid.

Claims (4)

637 218 1. Polymer matrix based on acrylamide for immobilizing antibodies in the radioimmunological determination of hormones and pharmaceuticals, characterized in that the polymer matrix consists of a copolymer of acrylamide. 2. Polymer matrix according to claim 1, characterized in that it consists of a copolymer of acrylamide and one or more of the compounds acrylic acid, methacrylic acid, methacrylamide, N-hydroxymethyl methacrylamide and N, N'-diallyltartardiamide and salts and esters of acrylic acid or methacrylic acid . 2nd PATENT CLAIMS 3. Polymer matrix according to claim 1 or 2, characterized in that it consists of a copolymer of acrylamide and 20 to 60 mol% methacrylic acid. 4. Polymer matrix according to one of claims 1 to 3, characterized in that it consists of a copolymer of acrylic acid and an alkali or alkaline earth metal salt of acrylic acid or methacrylic acid. The experience relates to a polymer matrix based on acrylamide for the immobilization of antibodies in the radioimmunological determination of hormones and pharmaceuticals. In the radioimmunological determination of hormones and pharmaceuticals, the hormones or pharmaceuticals react with an antibody. The specificity of the reaction depends on the immunological complementarity, i.e. there may be cross-reactivity with substances that have a similar chemical configuration. In the case of an estriol-Cs conjugate, the estriol is in an immunodeterminate configuration in which almost all the important functional groups of the estriol are exposed to the binding region of the antibody. Nevertheless, estriol conjugates, the sulfate or glucuronide residue of which are located at the phenolic C3, are bound to a considerable extent by the antibody. This is particularly disadvantageous for the determination of the free steroids if the conjugate concentration of the samples exceeds the steroid concentration by powers of ten, as is the case with the estrogens in the pregnant serum. For the above reasons, essentially methods have been used so far in which the specificity was ensured by solvent extraction before the radioimmunoassay. However, solvent extraction stands in the way of serial and machine sample processing. In order to simplify the radioimmunological methods using solid-phase techniques, it is known to enclose the antibodies in a gel. According to British patents 1452009 and 1452010, polyacrylamide is used as the gel to which N, N'-methylene-bis-acrylamide has been added, namely as a crosslinking agent, i.e. to give the gel the necessary pore size, density and strength. The advantages of the antibodies enclosed in such a gel are the exclusion of disruptive molecules of higher molecular weight, the saving of pipetting and centrifuging steps and the long shelf life of the antibody at room temperature. However, it has been shown that the binding specificity of the antibody, when enclosed in a polyacrylamide gel, does not meet the desired requirements.